Many soluble proteins, such as cytokines, hormones and growth factors, exert their functions by binding and activating cell-surface receptors (Arai, K.-I. et al. Annu. Rev. Biochem. 1990, 59, 783; Bazan, J. F. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6934; Ullrich A. and Schiessinger, J. Cell, 1990, 61, 203-212). These receptors are comprised of three distinct domains: an extracellular ligand-binding domain, a transmembrane domain and a cytoplasmic domain, which is responsible for signal transduction within the cell. Some receptors, such as those for erythropoietin (EPO), thrombopoietin (TPO), and granulocyte-colony stimulating factor (G-CSF), contain the ligand-binding and signal-transduction domains within the same polypeptide subunit. Others, such as receptors for interleukin-2 (IL-2), IL-3 and IL-6 have separate components for ligand-binding and signal transduction. Although the mechanism of receptor activation varies for specific receptor-ligand pairs, a common feature of many single-transmembrane receptors appears to be their aggregation on the cell membrane in response to binding of their specific ligands. This aggregation event can be in the form of homodimerization, in the case of receptors with a single subunit, or heterodimerization, in the case of receptors with different subunits. It has become clear that receptor aggregation is part of the biological signal by which the target cell responds to the presence of specific hormones and growth factors (Young, P. R. "Protein hormones and their receptors", Curr. Opin. Biotech. 1992, 3, 408-421; Heldin, C. H., "Dimerization of cell surface receptors in signal transduction). Typical examples of such receptors are growth factor receptors with tyrosine kinase activity as well as cytokine receptors.
Monoclonal antibodies have been discovered which have agonist activity to the dimeric receptors such as those from epidermal growth factor (EGF, Fernandez-Pol, J. J. Biol. Chem. 1985, 260,5003-11; Serrero, G. U.S. Pat. No. 5,723,115), G-CSF (Takahashi, T. et al. J. Biol. Chem. 1996, 271, 17555-17560), tumor necrosis factor (TNF, Fine, S. M. et al. J. Biol. Chem. 1996, 27126, 15303-15306.), growth hormone receptor (Rowlinson, S. W. et al. J. Biol. Chem. 1998, 2739, 5307-5314, EPO (Young, P. R. and Erickson-Miller, C. L. WO 9640231; Chaovapong, W. L. et al. WO 9748729.) and gp130, the common chain for members of the IL-6 family (Fourcin, M. et al. J. Biol. Chem. 1996, 271, 11756-11760). Ability of the monoclonal antibodies to activate the receptors is believed to be due to the presence of the two antigen binding sites, which can bridge the two receptor subunits and facilitate aggregation.
More recently peptides with agonist activity were identified by screening of phage display libraries against the EPO (Wrighton, N. C. et al. Science 1996, 273, 458-463; Wrighton, N. C. et al. U.S. Pat. No. 5,773,569) and TPO receptors (Cwirla, S. E. et al. Science 1997, 276, 1696-1699; Dower, W. J. et al. WO 9640750). The peptides ranged from 14 to 20 residues and activated the receptors by promoting their dimerization on the cell surface. These agonist peptides are unrelated to EPO and TPO and appear to act as dimeric agents, as demonstrated in the crystal structure of the EMPI/EBP complex (Livnah, O. et al. Science 1996, 273,464-471).
Despite the success of monoclonal antibodies and dimeric peptides in eliciting agonist response in certain dimeric receptors, they are not generally considered desirable candidates for development of pharmaceutical compositions. Lack of oral bioavailability and a limited serum half-life limit the desirability and efficacy of monoclonal antibodies and polypeptides as pharmaceutical agents. Consequently, a need exists for non-antibody ligands which have agonist properties towards dimeric cell-surface receptors.
Notwithstanding the fact that these receptors have been the subject of such research efforts for over a decade, only one application (PCT/US97/08,864) describes small organic molecules which exhibit agonist activity towards dimeric cell-surface receptors. This application does not mention zinc chelated small organic molecules.
As disclosed herein it has unexpectedly been discovered that zinc chelated receptor ligands have agonist properties towards dimeric cell-surface receptors.